X chromosome inactivation in female mammalian cells is an intriguing example of a '.developmentally-programmed system of genetic regulation. It is unusual in that it affects a large number of linked, but otherwise unrelated, genes, and requires differentiation between two copies of the same sequences within the same nucleus. The long-range goal of this work is to test the hypothesis that control of the time of replication of the active and inactive X chromosomes may play a critical role in the maintenance of their different transcriptional states. Two aspects of DNA structure that may be expected to be involved in this regulation, namely the control of replication initiation at origin sequences and differences in chromatin structure, are the primary emphases of this proposal. In particular, replication polarity methods and autonomous replication assays using EBV-based vectors will be used to identify transcription initiation sequences within a large region of the human X chromosome spanning the hypoxanthine phosphoribosyltransferase (HPRT) locus. The structural and functional characteristics of these sequences will be studied in detail and the potential for differential utilization of origin sequences in this region as a function of genetic activity will be examined. In addition, chromatin structure on the X chromosome will be studied at two levels. First, nuclear matrix association regions in the vicinity of the HPRT gene will be identified and their role in determining the topology of DNA loops on active and inactive chromosomes, as well as their relationship to replication control sequences will be established. Finally, pilot studies are proposed to ask whether different chromatin proteins are associated with active and inactive X chromosome sequences.